ABSTRACT: Signal transduction in the visual system involves a series of proteins that are sequentially activated via protein-protein interactions, thus passing the signal from the membrane receptor to a final target. The long- term objectives of this project are to elucidate the molecular mechanisms of activation and signal propagation in three proteins of the relay: rhodopsin, transducin and arrestin. It is now abundantly clear that these proteins are highly flexible. The flexibility involves ps-ns backbone fluctuations as well as a slower exchange between distinct conformational substates, both being central to function. Thus, mechanisms will not be revealed by structures determined in the confines of a crystal lattice, but will require new spectroscopic methods to complement crystallography and observe the proteins in action in a native-like environment. Toward this goal, one specific aim includes the development and application of new strategies in Site Directed Spin Labeling (SDSL). These include: (1) genetically encoded non-native amino acids for extending SDSL to previously inaccessible proteins and for attaching proteins to solid supports; (2) high pressure Electron Paramagnetic Resonance (EPR) for revealing protein flexibility and low-lying excited states and (3) pulse saturation recovery EPR and pressure-jump relaxation EPR for identifying conformational exchange and measuring exchange rates. Another specific aim is focused on application of these novel technologies together with Double Electron Electron Resonance (DEER) to elucidate structure and the role of dynamics in the activation of the proteins of signal transduction. Goals include a description of: (1) the conformational substates in activated rhodopsin (R*) and the mechanism of constitutive activation; (2) the conformational states of the transducin a-subunit and the structure of the R*-transducin complex and (3) the structure of the R*-arrestin complex, the transformations leading to the high-affinity binding form, and the mechanism of constitutive activation. A growing number of pathological conditions have been traced to genetic defects in the molecular switches of the visual and other signaling systems. In order to design pharmacological approaches for altering such pathological states, it is imperative to understand molecular mechanism involved in the signal relay. Success in this project is anticipated to contribute to this understanding.